Calnexin functions in antibacterial immunity of Marsupenaeus japonicus

Calnexin (Cnx) is an endoplasmic reticulum membrane–bound lectin chaperone that comprises a dedicated maturation system with another lectin chaperone calreticulin (Crt). This maturation system is known as the Cnx/Crt cycle. The main functions of Cnx are Ca²⁺ storage, glycoprotein folding, and quality control of synthesis. Recent studies have shown that Cnx is important in phagocytosis and in optimizing dendritic cell immunity. However, the functions of Cnx in invertebrate innate immunity remain unclear. In this research, we characterized Cnx in the kuruma shrimp Marsupenaeus japonicus (designated as MjCnx) and detected its function in shrimp immunity. The expression of MjCnx was upregulated in several tissues challenged with Vibrio anguillarum. Recombinant MjCnx could bind to bacteria by binding polysaccharides. MjCnx protein existed in the cytoplasm and on the membrane of hemocytes and was upregulated by bacterial challenge. The recombinant MjCnx enhanced the clearance of V. anguillarum in vivo, and the clearance effects were impaired after silencing MjCnx with RNA interference assay. Recombinant MjCnx promoted phagocytosis efficiency of hemocytes. These results suggest that MjCnx functions as one of the pattern recognition receptors and has crucial functions in shrimp antibacterial immunity.     

Modified expression of coagulation factor VIII by addition of a glycosylation site at the N terminus of the protein

Recently, it was shown that glycoproteins with N-glycans close to the NH₂ terminus can directly enter the calnexin/calreticulin cycle and bypass BiP binding. This should allow efficient secretion of glycoproteins such as factor VIII (FVIII) whose secretion is negatively affected by BiP interaction. Examination of the glycosylation pattern of the NH₂ terminus of FV and FVIII revealed N-glycans at positions 23 and 27 in FV and at position 41 in FVIII. To improve FVIII secretion, a 14-amino-acid-long polypeptide with (G3) or without (G0; control) three N-linked glycosylation consensus sites was inserted upstream of the NH₂ terminus of a B-domain deleted FVIII protein. Expression of G3- and G0-constructs in three different cell lines resulted in the same or even higher expression rate of protein as found for the B-domain deleted FVIII. However, as demonstrated by Western blot analysis, the G3- as well as the G0-protein variants were mainly retained inside the cells in similar amounts. Thus, glycosylation alone does not automatically lead to higher secretion rates, but must be in context to the normal structure of the FVIII protein. M. A. Srour and J. Grupp contributed equally to this work.     

The role of tapasin in MHC class I protein trafficking in embryos and T cells

Preimplantation mouse embryos express both classical (class Ia) and nonclassical (class Ib) MHC class I proteins, and yet are not rejected by the maternal immune system. Although the function of the embryonic MHC class Ia proteins is unknown, one MHC class Ib protein, Qa-2, the product of the preimplantation embryo development (Ped) gene, actually enhances reproductive success. Similar in structure to MHC class Ia proteins, Qa-2 protein is a trimer of the alpha (heavy) chain, β₂ microglobulin and a bound peptide. Studies on the folding, assembly and trafficking of MHC class Ia molecules to the cell surface have revealed this process to be dependent on multiple protein chaperone molecules, but information on the role of chaperone molecules in Qa-2 expression is incomplete. Here, we report the detection of mRNA for four chaperone molecules (TAP1, TAP2, calnexin and tapasin) in preimplantation embryos. We then focused on the role of the MHC-dedicated chaperone, tapasin, on Qa-2 protein expression. First, we demonstrated that tapasin protein is expressed by preimplantation embryos. Then, we used tapasin knockout mice to evaluate the role of tapasin in Qa-2 protein expression on both T cells and preimplantation embryos. We report here that optimal cell surface expression of Qa-2 is dependent on tapasin in both T cells and preimplantation embryos. Identification of the molecules involved in regulation of MHC class I protein expression in early embryos is an important first step in gaining insight into mechanisms of escape of embryos from destruction by the maternal immune system.     

Calnexin expression does not enhance the generation of MHC class I-peptide complexes

We investigated the requirement for calnexin in the biogenesis of MHC class I molecules. Mutant human cells lacking calnexin were infected with recombinant vaccinia viruses encoding mouse MHC class I molecules, K ^d , K^b , K^k , D ^d , D^b , and L^d . Flow cytometry indicated that each of the six MHC class I allomorphs was transported to the cell surface at similar rates in calnexin-deficient cells and transfectants expressing calnexin. For K^b and K ^d , the calnexin-independent biogenesis occurred regardless of whether the MHC class I molecules contained human or mouse β2-microglobulin. Also addressed was the effect of calnexin on the surface expression of K^b molecules bearing the immunodominant peptide from ovalbumin (OVA_{257 – 264} ). This was detected with a recently described monoclonal antibody specific for the K^b/peptide complex. Calnexin expression had no significant effect on the formation of K^b /peptide complexes generated from full-length OVA, cytosolic OVA_{257 – 264} , or endoplasmic reticulum-targeted OVA_{257 – 264} , which was expressed in the presence of the herpes simplex virus ICP47 protein to ensure detection of TAP-independent peptide-MHC class I complexes. Complementary results were obtained with TAP-independent formation of K ^d /peptide complexes. These findings indicate that calnexin is not required for the efficient assembly of MHC class I molecules with TAP-dependent or independent peptides.     

Calnexin/Calreticulin循环与糖蛋白内质网相关性降解

Calnexin/Calreticulin循环与糖蛋白内质网相关性降解 (GERAD)是真核细胞内负责糖蛋白折叠质量的重要监控机制 ,监督糖蛋白在内质网中空间构象的正确折叠 ,促进未完全折叠糖蛋白再折叠 ,错误折叠糖蛋白经GERAD途径被降解。内质网应激引起内质网功能障碍时 ,Calnexin/Calreticulin循环和 (或 )GERAD发生异常 ,错误折叠的糖蛋白在内质网堆积导致细胞功能障碍和某些疾病的发生。错误折叠糖蛋白从Calnexin/Calreticulin循环释放到GERAD一系列途径的分子机理现已逐渐明了 ,了解糖蛋白Calnexin/Calreticulin循环与内质网相关性降解过程对进一步设计针对病毒性肝炎、帕金森病等疾病的治疗方案有重要的指导意义。     

人脑胶质瘤中MAGE-D4和CANX mRNA的表达及其相关性分析

目的 探讨人脑胶质瘤中黑色素瘤相关抗原(MAGE)-D4和钙联蛋白(CANX)mRNA的表达水平,分析其相关性及临床意义.方法 应用基因表达谱交互式分析(GEPIA)数据库数据[胶质母细胞瘤(GBM)组织163例,低级别胶质瘤(LGG)组织518例,正常脑组织207例]分析MAGE-D4和CANX mRNA在人脑胶质瘤...     

内质网应激相关蛋白1对衣霉素诱导HepG2细胞内质网应激的影响

目的研究过表达内质网应激相关蛋白1(SERP1)对衣霉素诱导肝癌HepG2细胞内质网应激的影响。 方法以衣霉素诱导HepG2细胞发生内质网应激,将细胞分为以下5组：正常对照组、衣霉素组、衣霉素＋0.25μg SERP1转染组、衣霉素＋0. 5μg SERP1转染组和衣霉素＋1.0μg SERP1转染组,每组实验重复3次;采用MTT法检测不同浓度与作用时间的衣霉素对HepG2细胞存活率的影响,以吸光度(A)值表示。Western blot法检测各组细胞内内质网应激标志蛋白葡萄糖调节蛋白(GRP78)、C/EBP同源蛋白(CHOP)以及钙联蛋白的表达水平。采用SPSS 15.0统计软件进行统计学分析,比较蛋白表达水平。 结果与对照组相比,衣霉素处理组HepG2细胞中内质网应激标志性蛋白GRP78、CHOP及Calnexin蛋白表达量显著升高,分别为对照处理组的3.8倍(t＝11.5,P<0.05)、1.3倍(t＝3.498,P<0.05)和1.4倍(t＝4.1,P<0.05),差异均有统计学意义;随着SERP1过表达量的逐渐升高,变化呈现剂量依赖性。随着SERP1转染剂量的增加,各组GRP78蛋白的表达较单独衣霉素处理组分别下降了12%[(1.83±0.29)A值,(1.61±0.13)A值,t＝2.36,P>0.05]、24%和30%[(1.83±0.29)A值,(1.40±0.11)A值,(1.27±0.21)A值;F＝50.56,P<0.05],CHOP蛋白的表达水平分别下降了23%, 29%和34%[(1.0±0.15)A值,(0.79±0.07)A值,(0.72±0.55)A值,(0.67±0.14)A值;F＝9.532,P<0.05],Calnexin蛋白的表达水平分别下降了5%[(1.20±0.18)A值,(1.15±0.13)A值;P>0.05]、24%和28%[(1.20±0.18)A值,(0.92±0.07)A值,(0.87±0.18)A值;F＝8.116,P<0.05]。 结论外源性过表达SERP1蛋白通过下调内质网应激蛋白的表达,降低HepG2细胞内质网应激水平,缓解内质网应激介导的细胞损伤。     

沉默Calnexin基因表达对胃癌细胞SGC-7901内质网应激凋亡及其信号通路的影响

目的探讨沉默钙连蛋白（Calnexin）基因对胃癌细胞SGC-7901内质网应激凋亡信号通路的影响。方法构建靶向Calnexin基因的慢病毒干扰载体,利用包装细胞293T获得重组慢病毒,转染人胃癌SGC-7901细胞,并分为转染shRNA-Calnexin的实验组,转染shRNA空白质粒的空白载体组,并将未转染的SGC-7901细胞作为对照组。应用RT-PCR技术检测靶向沉默Calnexin的表达,应用四甲基偶氮唑蓝（MTT）检测各组细胞增殖情况;流式细胞术检测各组细胞凋亡情况;Westernblot检测GRP94、IRE1、ATF6及CHOP表达水平。结果转染shRNA沉默Calnexin基因可明显下调CalnexinmRNA的表达,实验组与对照组比较差异有统计学意义（P＜0.05）;MTT法检测实验组细胞转染后细胞增殖能力明显下降（P＜0.05）;沉默Calnexin基因可提高细胞凋亡率,转染72h后实验组与对照组比较,差异有统计学意义（P＜0.05）;沉默Calnexin基因使胃癌细胞中GRP94、IRE1、ATF6及CHOP蛋白表达水平明显升高（均P＜0.05）。结论靶向沉默Calnexin基因抑制人胃癌细胞增殖和促进细胞凋亡可能是通过抑制内质网应激信号通路实现的。